Endosomal localization of the serum resistance-associated protein in African trypanosomes confers human infectivity

Abstract

Trypanosoma brucei rhodesiense is the causative agent of human African sleeping sickness. While the closely related subspecies T. brucei brucei is highly susceptible to lysis by a subclass of human high-density lipoproteins (HDL) called trypanosome lytic factor (TLF), T. brucei rhodesiense is resistant and therefore able to establish acute and fatal infections in humans. This resistance is due to expression of the serum resistance-associated (SRA) gene, a member of the variant surface glycoprotein (VSG) gene family. Although much has been done to establish the role of SRA in human serum resistance, the specific molecular mechanism of SRA-mediated resistance remains a mystery. Thus, we report the trafficking and steady-state localization of SRA in order to provide more insight into the mechanism of SRA-mediated resistance. We show that SRA traffics to the flagellar pocket of bloodstream-form T. brucei organisms, where it localizes transiently before being endocytosed to its steady-state localization in endosomes, and we demonstrate that the critical point of colocalization between SRA and TLF occurs intracellularly.

Fig. 1.

SRA traffics to the flagellar pocket. (A) Diagram of SRA, indicating the predicted positions of the N-terminal signal peptide (SS), GPI anchor addition site (D388), C-terminal peptide (gray), and Ty epitope tag. (B and C) SRA traffics to the flagellar pocket. Live cell binding studies at 3°C with Alexa Fluor 594-transferrin (red) (B) or Alexa Fluor 594 anti-Ty (SRA) (red) (C). After binding and fixation, basal bodies were stained with anti-YL1/2 (green) (B), and the paraflagellar rod was stained with anti-PFR (green) (B and C). The white bracket shows the flagellar pocket region, and the arrowheads denote the positions of the basal bodies. (D and E) Cells were fixed under nonpermeabilizing (D) and permeabilizing (E) conditions and stained for SRA with anti-Ty (green). Cells were also stained for the lysosomal compartment with anti-TbCatL (red). Image acquisition was carried out at the same exposure, and images were contrasted to the same extent.

Fig. 2.

The initial interaction between SRA and TLF does not take place at the flagellar pocket. (A) Binding of Alexa Fluor 594–anti-Ty (SRA) (red) in the flagellar pocket of T. brucei brucei 060^R SRA-Ty cells. Anti-PFR staining is shown in green. (B and C) Binding (B) and uptake (C) of Alexa Fluor 488-TLF (green) in T. brucei brucei 221 (TbHpHbR⁽⁺⁾), T. brucei brucei 060^R (TbHpHbR⁻), and T. brucei brucei 060^R SRA-Ty (TbHpHbR⁻) cells. White arrowheads show binding in the flagellar pocket. DIC, differential interference contrast microscopy. (D) Flow cytometry analysis of Alexa Fluor 488-TLF uptake. Strains used included T. brucei brucei 221 (TbHpHbR⁺), T. brucei brucei 060^R (TbHpHbR⁻), and T. brucei brucei 060^R SRA-Ty (SRA-Ty/TbHpHbR⁻).

Fig. 3.

Endosomal localization of SRA. (A to C) Western blot (A) and immunofluorescence staining (B and C) with anti-HA (Rab5a) (red) of T. brucei brucei 221 TbRab5aHA transfectants. Lysosomal (anti-TbCatL) staining is shown in green. (D to G) Binding and uptake studies using T. brucei brucei 221 TbRab5aHA (D and E) and T. brucei brucei 221 SRA-Ty (F and G) transfectants. Live cell binding results of Alexa Fluor 594-transferrin (red) at 3°C, 0 min (D and F), followed by temperature shift to 37°C for 1 min then fixation with 1% paraformaldehyde to halt vesicle trafficking (F and G) are shown. Postfixation, TbRab5aHA was stained with anti-HA (green) (D and E), and SRA was stained with anti-Ty (green) (F and G). (H) Steady-state localization of SRA-Ty and TbRab5aHA. Fixed, permeabilized T. brucei brucei 221 TbRab5aHA;SRA-Ty transfectants were stained with anti-Ty (SRA; green) and anti-HA (Rab5a endosomes; red). (I) Uptake of Alexa Fluor 488-TLF (green) in T. brucei brucei 221 TbRab5aHA transfectants. Rab5a endosomes are shown by the anti-HA (red) staining. The nucleus and kinetoplast were localized by DAPI staining. White arrowheads indicate colocalization.

Fig. 4.

Loss of the GPI anchor disrupts flagellar pocket localization. (A) Schematic diagram of SRA-Ty and SRAΔGPI. (B) Western blot of SRA-expressing T. brucei brucei transfectants probed with anti-Ty (SRA) and anti-La (La protein loading control). (C and D) Binding of Alexa Fluor 594 anti-Ty (SRA) antibody at 3°C in SRA-Ty cells (C) and SRAΔGPI cells (D) is shown in red. PFR staining is shown in green. (E and F) SRA-Ty (E) and SRAΔGPI (F) cells were stained for SRA (green) and lysosomal localization (red).

Fig. 5.

Localization of SRA to the flagellar pocket is not critical to resistance. (A and B) Uptake of Alexa Fluor 488-TLF (green) at 37°C for 1 min postbinding in SRA-Ty (A) and SRAΔGPI (B) cells. SRA was visualized via anti-Ty staining (red). White arrowheads indicate colocalization. (C) Untransfected T. brucei brucei 221, T. brucei brucei 221 SRA-Ty, and T. brucei brucei 221 SRAΔGPI cells were incubated with increasing concentrations of purified TLF-1. Percent lysis was determined after 2 h at 37°C.

Fig. 6.

SRA is rapidly turned over by lysosomal proteolysis. Immunofluorescence staining of fixed, permeabilized T. brucei brucei 221 SRA-Ty transfectants is shown. (A to C) Cells were untreated (A) or FMK024 treated (B and C) prior to fixation (20 μM; 37°C, 1 h). Anti-Ty (SRA; green) and anti-TbCatL (red) staining showed SRA and lysosomal localization, respectively. Image acquisition was carried out at the same exposure, and images were contrasted to the same extent. (D) Anti-Ty (SRA) Western blot of untreated and FMK024-treated T. brucei brucei 221 SRA-Ty transfectants, showing accumulation of SRA due to protease inhibition. (E to G) Uptake of Alexa Fluor 488-TLF (green) in T. brucei brucei 221 (E) and T. brucei brucei 221 SRA-Ty (F and G) cells. Cells were either untreated (E and F) or treated with FMK024 (20 μM; 37°C, 1 h) (F) during TLF-1 endocytosis. Anti-TbCatL (lysosomal) staining is shown in red. The nucleus and kinetoplast were localized by DAPI staining. White arrowheads indicate colocalization.